PROJECT SUMMARY/ABSTRACT Oxidative injury is a common injury seen in many diseases and conditions, including neurodegenerative diseases and aging. In order to dissect the mechanisms that protect cells against oxidative injury, we started by generating a unique Drosophila melanogaster strain that can tolerate severe, normally lethal, hyperoxic conditions through laboratory selection by gradually elevating oxygen levels (i.e., the hyperoxia-selected flies (HOF)). By comparing nave control with HOF flies, we have identified differences in genomic DNA sequences as well as gene and protein expression that regulate responses to oxidative stress and injury. Importantly, we have confirmed experimentally the role of a subset of these candidate genes in protecting against oxidative injury. The majority of the differentially expressed mitochondrial proteins were found to be the modifiers of Notch signaling. Furthermore, we found that the kst gene is under strong selection in the HOF populations (p<0.01, McDonald-Kreitman test), and its expression was significantly up-regulated in the HOF flies. We therefore hypothesize that Notch signaling and the human ortholog of kst (i.e., the spectrin gene family) regulate cellular tolerance to oxidative stress in humans play a critical role in regulating cell survival or susceptibility to oxidative insults in neuronal and glial cells. In the current application, we will translate our findings from Drosophila into humans by elucidating the role of Notch pathway as well as the kst ortholog genes in regulating cell survival under oxidative stress using human iPSC-derived neurons and glia. Both hyperoxia and paraquat will be used to generate oxidative stress in these human cells. Our specific aims are 1) to elucidate the role of Notch in regulating oxidative responses in human iPSC-derived neuronal precursor cells and differentiated neurons and glial cells; and 2) To study the role of human orthologs of the Drosophila kst gene in oxidative tolerance or susceptibility in human neurons and glial cells. The overall goal of the current project is to identify novel therapeutic targets for the treatment and prevention of oxidative stress-induced brain injury.